Recent technological advances that allowed for the mapping of selected RNA modifications on a transcript-wide scale have revealed a wide-spread distribution of N6-methyladenosine (m6A), pseudouridine (Ψ), 5- methylcytosine (m5C), ribose 2’-O-methylation (Nm), and adenosine-to-inosine (A-to-I) RNA editing on mRNA. The m6A is the most abundant base modification in RNA (over 118,000 m6A peaks in human transcriptome), and controls a plethora of systems, including stem cell proliferation and differentiation, cellular heat shock responses, spermatogonia differentiation, maternal-to-zygotic transition, X-chromosome inactivation, UV DNA damage response, neurogenesis, and tumorigenesis. A variety of illnesses have been further linked with m6A methylation disorder, including acute myeloid leukemia, gliomas, breast cancer, and lung cancer in humans. Despite widespread recognition of the important functions of RNA modifications, the availability of technologies that can monitor and image site-specific RNA modifications are lacking. Novel tools for the site-specific detection of RNA modifications in cells and other biological samples are urgently needed. The overall goal of this project is to develop novel, easy to use fluorescence-based methods and reagents for sequence specific imaging of RNA modifications. The goal of Phase I was to establish the feasibility of novel m6A antibody-based imaging reagents. In Phase I, we have developed the first-in-class and the best-in-class homogeneous antibody-based novel assays to determine the extent of site-specific m6A RNA modifications. Furthermore, we greatly exceeded the original goal of Phase I by developing an additional novel “no antibody” method which entirely avoids the use of m6A antibody, reducing reagent’s complexity and cost. With these two novel methods established in Phase I, we are ready to move into a Phase II project. Our aims include Aim 1. Develop optimal protocols for staining fixed cells using the methods established in Phase I. Aim 2. Test methods to increase sensitivity of detection. Aim 3. Evaluate and compare the performance of all method variants using cell culture systems. Aim 4. Expand and validate the methods to measuring other RNA modifications such as A- to-I RNA editing and m5C. In addition, all the methods, reagents, and protocols developed and validated internally by Mediomics will be sent to a leading laboratory in this field for third party validation to ensure that Mediomics' newly developed assays work well in any laboratory that may have an interest in epitranscriptomics and the quantification of RNA modifications. Successfully validated assay kits will be ready for commercialization. These novel methods could become the first standard approach for analyzing RNA modifications in any sample and will enable researchers to perform fluorescence-based imaging for RNA modifications in important biological samples, including brain and cancer samples. It may further serve as an important ...